The inotropic activity of digitalis genins is dependent upon C(17) side-group carbonyl oxygen position.

The inotropic potencies of four digitalis genins were studied utilizing cat left atrial strips. The genin concentration required to induce a 50% increase of isometric tension (T50) was found to closely correlate with the degree of displacement (D) of the C(17) side-group carbonyl oxygen from the position of that atom in digitoxigenin. The line of regression was: log T50 = 0.54D - 6.85, r2 = 0.98, P less than 0.008. These observations were related to recently reported cat ventricular Na+, K+ -ATPase inhibitory potencies of the same genins [expressed as 50% inhibitory (I50) concentrations]. I50 correlated strongly with T50: log I50 = 0.78 log T50 - 1.68, r2 = 0.99, P less than 0.003. Thus, the activity of digitalis genins towards their receptor in intact cardiac tissue is closely related to genin carbonyl oxygen position as well as to Na+, K+ -ATPase inhibitory activity. These results further support our earlier conclusions, based upon isolated Na+, K+ -ATPase studies, that the digitalis genin C(17) side-group carbonyl oxygen position versus activity relationship is biologically relevant and may prove to be a useful unifying structural model in the further elucidation of the mechanism of digitalis-receptor interactions.

Cardiac glycosides. 1. A systematic study of digitoxigenin D-glycosides.

A series of digitoxigenin glycosides was studied: five with beta-D-sugars varying stepwise in sugar structure from beta-D-digitoxose to beta-D-galactose, including one beta-D/alpha-D pair. I50 values for these glycosides and digitoxigenin were determined with hog kidney Na+, K+-ATPase. These data suggest a major and unexpected role for 4'-OH conformation in the sugar. All the glycosides with an equatorial 4'-OH were more active than the two with the 4'-OH axial [digitoxigenin beta-D-galactoside (6) I50 = 6.45 X 10(-8) M; digitoxigenin 2'-deoxy-alpha-D-ribo-hexopyranoside (alpha-3a) I50 = 9.33 X 10(-8) M; digitoxigenin I50 = 1.17 X 10(-7) M]. Stereochemistry of the 3'-OH had much less of an activity role than that of the 4'-OH, in contrast to existing models of "sugar-site" binding.

Photoaffinity labeling of the sodium- and potassium-activated adenosinetriphosphatase with a cardiac glycoside containing the photoactive group on the C-17 side chain.

The synthesis and properties of a radiolabeled glycoside photoaffinity probe, [3H]-(3 beta,5 beta,14 beta, 20E)-24-azido-3-[(2,6-dideoxy-beta-D-ribo-hexopyranosyl) oxy]-14-hydroxy-21-norchol-20(22)-en-23-one, containing the photoactive group at the C-17 side chain of the steroid moiety are reported. The molecule binds to the sodium- and potassium-activated adenosinetriphosphatase from porcine kidney outer medulla under type II binding conditions [5 mM MgCl2, 3 mM phosphate, 2 mM ethylenediaminetetraacetic acid, 30 mM tris(hydroxymethyl)aminomethane, pH 7.2, 37 degrees C] in the dark with an equilibrium dissociation constant of (1.4 +/- 0.3) X 10(-7) M. Ultraviolet irradiation of a solution of enzyme plus 3H-labeled probe, followed by analysis of covalently incorporated radiolabel, shows ouabain-displaceable labeling exclusively of the alpha subunit of the sodium- and potassium-activated adenosinetriphosphatase. These data indicate that the binding site of the C-17 side group of cardiac glycosides is located on or near the alpha subunit of this enzyme.

Interaction of (Na+,K+)-ATPases and digitalis genins. A general model for inhibitory activity.

Previous models of digitalis genin interaction with the (Na+,K+)-ATPase system (the putative receptor for such drugs) were deficient in explaining the (Na+,K+)-ATPase inhibitory activity of a number of digitalis genin analogues. With rat brain (Na+,K+)-ATPase we observed that the C-17 side chain carbonyl (C = O) oxygen distance of a given genin in relation to its position in the reference compound digitoxigenin was the primary determinant of its biological activity. With a number of genin analogues, we observed a strict correlation of this structural parameter with its binding site compatibility as well as inhibitory potency with respect to the (Na+,K+)-ATPase. In every case the correlation to inhibition data was obtained using a minimum energy conformation for the genin structure. The general applicability of that model is now proposed based on the following observations. The carbonyl oxygen position versus the biological activity relationship fully holds with (Na+,K+)-ATPase preparations from other tissues and species and also when different binding conditions are used for the enzyme genin interaction. The relationship is equally valid for the K+-p-nitrophenyl phosphatase activity. Correlations of the data obtained under these various conditions provide further support for this relationship and for the concept that altered affinities of the enzyme for a given genin under different binding conditions reflect conformational variations of a single binding site.